Technical Abstract:
The enzyme alternanase, produced by Bacillus sp. NRRL B-21195, hydrolyzes alternan, a polysaccharide produced by certain strains of Leuconostoc mesenteroides that consists of glucose linked by alternating alpha(1-6), alpha(1-3) linkages. The main product of enzymatic hydrolysis by alternanase is a novel cyclic tetrasaccharide of glucose that also has alternating linkages between the glucose moieties. An improved purification scheme for alternanase has been developed that incorporates the use of isomaltosyl units linked to agarose for selectively binding the alternanase enzyme. The purification procedure is relatively simple and rapid and can be scaled to permit large quantities of enzyme to be purified in high yield; 1 ml of resin was capable of bind 2 mg of alternanase. Affinity chromatography on immobilized isomaltose yielded two protein bands when examined by SDS-PAGE, which could be separated by preparative isoelectric focusing, resulting in highly purified alternanase with a 33% yield. Alternanase is also capable of converting the trisaccharide panose into the cyclic tetrasaccharide with the concomitant release of glucose; a coupled enzyme assay that utilizes glucose oxidase to measure the release of glucose enables a direct measurement of alternanase activity.